The flavivirus capsid protein (C) is a positively charged protein that presumably forms nucleocapsid structures when complexed with virion RNA. At least two forms of capsid exist, that which results from initiation of translation at the most amino- terminal Met in the long ORF in genomic RNA (Cap1) and that which results from initiation of translation at the next downstream Met codon (Cap2). The dengue virus type 4 (DEN4)C can be detected in the nuclei of LLCMK2 cells infected by DEN4 virus. Although flaviviruses are generally thought to replicate in the cytoplasm of infected cells, this result suggests a nuclear involvement in these processes. Proteins that are actively transported from the cytoplasm to the nucleus contain a nuclear localization signal (NLS). The DEN4 C contains several candidate NLS's but the best fit occurs within the 13-amino- acid amino-terminus of Cap1 not included in Cap2. Thus, one would predict that the DEN4 Cap1 but not Cap2 would be transported to the nucleus. To formally identify the NLS in the DEN4 C, Cap1 and Cap2 cDNA were cloned into a vaccinia virus-based expression vector, pTM-1. Using this expression system, we have thus far demonstrated nuclear localization of Cap1 in LLCMK2 cells by IFA in a distribution indistinguishable from that obtained after DEN4 virus infection. Nuclear localization of Cap2 has thus far not been demonstrated, in keeping with our original hypothesis. Several mutations of the putative NLS in Cap1 have also been generated using PCR. Nuclear localization of these Cap1 mutants remains to be determined but should lead to definitive identification of the NLS. We then plan to assess the requirement for and the effects on the cell of nuclear localization of C during DEN4 virus infection by replacing wild-type C sequences in a full-length genomic cDNA clone with NLS-minus mutant C sequences. Mutant full-length cDNA will be used to generate infectious RNA transcripts in vitro. The phenotype of mutant virus arising from transfection with such transcripts can then be assessed. The effects of C on nuclear function can be assessed using either infectious RNA transcripts or by expression of C using the vaccinia-based system.